Medicament for treating tumours and their metastases

ABSTRACT

The invention relates to a medicament against tumors and their metastases, which preferentially settle in bone tissue, comprising as active ingredient at least one binding molecule which binds to bone sialoprotein or a fragment thereof in serum or plasma.

This application is the National Stage of International ApplicationPCT/EP02/06456 filed on Jun. 12, 2002, to which priority is claimedunder 35 U.S.C. §120, which in turn claims priority to both of EuropeanApplication 01114388.0, filed on Jun. 13, 2001 and German Application10128639.2, filed on Jun. 15, 2001, under 35 U.S.C. §119.

FIELD OF THE INVENTION

The invention relates to medicaments for the treatment and combating oftumors and of metastases, which particularly frequently settle in bonytissue.

BACKGROUND OF THE INVENTION

At the present time many medicaments are in development which areintended to combat tumors and their metastases which spread in bone.Despite all advances in medicaments, bone metastases, however, do notcount as being curable or treatable. The are attempts, by means ofantibodies against surface antigens of tumor cells, to combat theirmetastases. Bone metastases are, however, despite this the cause ofdeath in 73% of cases of tumors of the breast, and 68% of cases fortumors of the prostate gland. For tumors of other tissues the followingfigures apply: cervix 50%, thyroid gland 42%, bladder 40%, lungs 36%,ovaries 9% and colon 6%.

Tumor cells which express the so-called bone sialoprotein have thepeculiarity that they prefer to settle in bony tissue and buildmetastases there, particularly in the case of tumors of the prostategland, breast, lungs, kidney and thyroid and less frequently in the caseof malign and semi-malign tumors. The bone sialoprotein (BSP) is aphosphorylated bone glycoprotein having a relative mass of ca. 80 kDa inthe SDS-PAGE. The DNA for BSP codes for a peptide sequence of ca. 33 kDa(Fisher (L. W. et al. (1990), J. Biol. Chem. 265, 2347-51; U.S. Pat. No.5,340,934). BSP is one the few matrix proteins the occurrence of whichon mineralising tissue such as bones, dentin and calcifying cartilage isrestricted. BSP represents ca. 10 to 15% if the total non-collagenicproteins in the bone matrix. It is as a rule expressed by cells whichtake part in the formation of dentin, bones and cartilage, for exampleosteoblasts, developing osteocytes, hypertrophic chondrocytes,odontoblasts and cementoblasts, but also by the trophoblasts in theplacenta and some types of cancer cells, e.g. in the case of lungs,breast, prostate, kidney, thyroid and neuroblastoma primary andsecondary tumors, in the case of multiple myeloma and in bonemetastases. The degree of expression of BSP by the tumor closelycorrelates with the severity of the cancer (Waltregny D. et al.,Increased expression of bone sialoprotein in bone metastases comparedwith visceral metastases in human breast and prostate cancers, in J.Bone Miner. Res., 2000, 15(5), 834-43; Bellahcène, A. et al., Bonesialoprotein expression in primary human breast cancer is associatedwith bone metastases development, in J. Bone Miner. Res., 1996, 11,665-670; Waltregny, D. et al., Prognostic value of bone sialoproteinexpression in clinically localised human prostate cancer, in Journal ofthe National Cancer Institute, 1998, 90, 1000-1008; Bellahcène, A. etal., Expression of bone sialoprotein in primary breast cancer isassociated with poor survival, in Int, J. Cancer, 1996, 69, 350-353).

BSP, as an adhesion molecule, is supposed to bring about attachment anddissemination of cells on the tissue matrix, since in vitro it formscrystallisation nuclei for biological apatite and in vivo takes part inmineralisation. The switching off of the BSP gene in knock-out miceleads to no recognisable disruption of the building and functioning ofthe skeleton. In tumors BSP is attributed with participation inmicrocalcification (Castronovo, V. et al., Evidence that breast cancerassociated microcalcifications are mineralized malignant cells, in Int.J. Oncol., 1998, 12, 305-308) and the colonisation of bones bymetastasising tumor cells (Bellahcène, A. et al., Expression of bonesioloprotein in primary breast caner is associated with poor survival,in Int. J. Cancer, 1996, 69, 350-353).

The level of concentration of BSP in the serum of patients with primarycarcinomas serves for diagnosis of whether these patients have bonemetastases or such are likely to arise from the primary tumor (DiplomaThesis of Ms. Ina-Alexandra Meier, Development of a radioimmunoassay forthe determination of bone sialoprotein (BSP) [“Entwicklung einesRadioimmunoassays zur Bestimmung von Bonesialoprotein (BSP) ]”, 1996,Darmstadt, Technical University [Fachhochschule], Specialist FieldChemical Technology [FB Chemische Technologie]; Dissertation of Mr.Markus Karmatschek, Isolation of bone sialoprotein from human bones,Structure of a radioimmunoassay for the measurement thereof in serum[“Isolierung von Bonesialoprotein aus humanem Knochen, Aufbau einesRadioimmunoassays zur dessen Messung im Serum”], 1996; Specialist Fieldof Biology of the Technical University of Darmstadt [FB Biologie derTechnischen Hochschule Darmstadt]; Diel I. J. et al., Elevated bonesialoprotein in primary breast cancer patients is a potent marker forbone metastases; in Proceedings of ASCO, 1998, 17, Abstract 461; Diel I.J. et al, Serum bone sialoprotein in patients with primary breast canceris a prognostic marker for subsequent, bone metastasis, in Clin. CancerRes., 1999, 5, 3914-19; DE 198 13 633; DO 198 21 533; WO 99/50666).

However, in body fluids free BSP is bound by complement factor H withhigh affinity. Further, BSP can bind to various receptors. Thus, therehave been produced in rabbits antibodies against various peptide partialstructures of BSP (Fisher, L. W. et al., Antisera and cDNA probes tohuman and certain animal model bone matrix noncollagenous proteins. ActaOrthop Scand Suppl., 1995, 266, 61-655), against recombinant BSP (StubbsJT 3^(rd) et al., Characterization of native and recombinant bonesialoprotein: delineation of the mineral-binding and cell adhesiondomains and structural analysis of the RGD domain. J. Bone Miner. Res.1997 12(8), 1210-22), and against BSP isolated from bones, whichantibodies failed to recognise any BSP in human serum. The larger factorH molecule of 150 kDa probably masks the smaller BSP (of ca. 65 kDa), sothat antibodies cannot bind. Further, factor H is present in excess inthe serum (0.5 mg factor H/mL in comparison to BSP with <20 ng/ml Serumin the case of healthy persons and max. 160 ng/ml in the case of tumorpatients). It has been asserted that immunological direct determinationof BSP in body fluids is impossible, without reducing samplepreparation, due to the binding to the factor H and possibly thattrophoblasts and BSP producing tumor cells are thereby protected fromattack by the immune system, since the factor. H belongs to thecomplement system and is known to bring about a restriction of thealternative pathway to complement lysis (Fedarko N. S. et al., Factor Hbinding of bone sialoprotein and osteopontin enables tumor cell elvasionof complement-mediated attack, in J. Biol. Chem., 200, 275, 16666-16672;WO00/062065). Further, BSP can specifically bind to the integrinreceptors on the cell surface through its own recognition, sequence(arginine-glycine-aspartate, RGD). In the case of expression of BSP thetumor cells are then supposed to bind the factor H in the blood and inthe tissue fluids to their cell surfaces, or concentrate it around them.Such a protection of BSP from the complement system of the blood of themother is suspected also for the trophoblasts in the placenta (FedarkoN. S. et al. Factor H binding of bone sialoprotein and osteopontinenables tumor cells evasion of complement-mediated attack, in J. Biol.Chem., 200, 275, 16666-16672; WO 00/062065). Further there is alsosuspected a function of BSP in angiogenesis. Along with the adhesion ofosteoclasts and osteoblasts to the bone matrix—through the binding ofthe RGD recognition sequence in the matrix to the alpha(v)beta(3)integrin receptors on the cell wall—it is also observed that theadhesion, dissemination and orientation of the endothelial cells isprobably mediated by BSP. Namely, blood vessel formation around a tumoroccurs in parallel with the BSP expression in the tumor cells(Bellahcène A et al., Bone sialoprotein mediates human endothelial cellattachment and migration and promotes angiogenesis, in Circ. Pes. 2000,86(8), 885-91).

These characteristics thus make BSP a starting point for medicaments ofall kinds. Thus, the binding of BSP via the RGD sequence to vitronectinor integrin receptors of tumor and epithelial cells can be restricted byantagonists (U.S. Pat. No. 6,069,158; U.S. Pat. No. 6,008,213; U.S. Pat.No. 5,849,865; van der Pluijm et al., Bone Sialoprotein peptides arepotent inhibitors of breast cancer cell adhesion to bone in vitro, inCancer Res. 1996, 56, 1948-1955) EP 1 084 719 A1 teaches apharmaceutical composition having BSP as active substance for thesupport of the repair of damaged bone and connective tissue. WO 94/13310teaches a composition having a BSP binding protein of staphylococcusaureaus as active ingredient. WO 00/36919 discloses regulatory elementsfor the purposive monitoring and suppression of the expression of BSP intumor and connective tissue cells, which promote calcification. Thus,generally the substances of the regulation of the cell growth and cellmigration are of particular interest from a diagnostic and therapeuticpoint of view. There are, however, still very many unknown factors whichcontrol cancer growth, whereby primary and secondary tumors andcolonised organs interact. Here, important steps are the invasion,adhesion, migration and cell division of the tumor cells. Along withmatrix metalloproteinases, adhesion molecules and chemotactic factorsplay a particular role. A medicament for combating and also for healingbone metastases on the basis of antibodies and binding molecules againstBSP is not known. It is also not known that the BSP of tumor cells isdifferent from the BSP of normal healthy cells.

SUMMARY OF THE INVENTION

The subject of the invention is a medicament for therapy of tumors andtheir metastases, which preferentially settle in bony tissues, includingas active ingredient at least one binding molecule which binds to bonesialoprotein or a fragment thereof in serum or plasma. The activeingredient is preferably an antibody or an aptamer or spiegelmer(Noxxon, Berlin, Germany), on a DNA or RNA basis and further binds amolecule which corresponds to a bone sialoprotein modified chemically ornaturally in its glycosylation. The binding molecule may be an antibodyor an aptamer which specifically binds bone sialoprotein from tumorcells, or also the binding structure of natural BSP receptor or thefactor H molecule. The binding molecule binds to or it can be producedagainst bone sialoprotein from bone material, which is modified in itsglycosylation, the donor of which was not capable of normalglycosylation of bone proteins.

In a particularly preferred embodiment, the medicament contains asactive ingredient an antibody or a plurality of antibodies against humanbone sialoprotein (hBSP), whereby the antibodies bind epitopes which arepresent on human sialoprotein from tumor cells, the post-translationalglycosylation of which is modified or incomplete, in comparison withnormal bone sialoprotein from bones, in the region of the amino acids120 to 135 (SWISSPROT: SIAL_HUMAN, Acc. No. P21815, incl. signalsequence) containing the amino acids TGLAA. The medicament in accordancewith the invention may also contain as active ingredient an antibodyand/or an aptamer produced against a hBSP epitope, including the aminoacid sequence TGLAA (SEQ ID NO: 15) or YTGLAA (SEQ ID NO: 16) andoptionally sugar groups and a carrier molecule. The active ingredient ispreferably a chicken IgY antibody. The chicken IgY antibody may also bea corresponding human or humanised antibody. Further preferred aremedicaments whereby the binding molecule contains as a bispecificantibody also an additional paratope which is preferably specific forepitopes of CD3. The active ingredient may also be an immunotoxin thatis a conjugate of binding molecule and a residue having cytotoxicactivity. The immunitoxin may for example be a conjugate which containsthe ricin-A-chain or a non-binding fragment of the diphtheria toxin. Thebinding molecule may, further, be coupled with a radionuclide so thatthe medicament can also be put to use for immune scintigraphy or for thelocalisation and observation of development of bone metastases.

The medicament in accordance with the invention may also contain atleast one antibody, ligand or inhibitor, from the group comprisingadhesion molecules, membrane associated proteases, receptors whichmediate chemotaxis, chemokine receptors, apoptosis inducing substances.The inhibitors can be so selected that they at least partially block BSPand modulate its function. In terms of its range of applications, themedicament in accordance with the invention is thus particularlysuitable for the treatment of tumors from the group comprising prostate,breast, lung, kidney and thyroid tumors, tumor diseases of the bloodsystem, of the lymphatic system, and of the heart and circulatorysystem, the nervous system, the respiratory tract, the digestive tract,the endocrinic system, the skin including adnexa, the locomotory systemand of the urogenital tract.

There will now be described further features and advantages of theinvention with reference to the examples and the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

There is shown:

FIG. 1 a Western Blot with tumor and bone specific isoforms of BSP;

FIG. 2 a Western Blot of the cell culture supernatant of non-transfectedEBNA-293 cells (negative control) and transfected EBNA-293 cells havingthe expression constructs GST-EK-BSP and his₆-myc-EK-BSP with theemployment of a monoclonal mouse anti-BSP antibody;

FIG. 3 the amino acid sequence of secreted BSP (SEQ ID No. 2) accordingto Fisher et al. (1991);

FIG. 4 a a curve indicating lesion size in square millimeters of a bonemetastasis in the tibia of rat 988 over the observation and therapyperiod;

FIG. 4 b an X-ray image 31 days post-OP of the lesion in the tibia,before the commencement of therapy;

FIG. 4 c an X-ray image 52 days post-OP of the lesion with stillcontinuing lysis of the bone, after the commencement of therapy;

FIG. 4 d X-ray 73 days post-OP of the regressing lesion;

FIG. 4 e X-ray 126 days post-OP of the healed lesion;

FIG. 4 f CT reconstruction of the lesion 31 days post-OP;

FIG. 4 g CT reconstruction of the regressing lesion 80 days post-OP;

FIG. 5 a curve of lesion size, in square millimeters, of a bonemetastasis on the distal femur of rat 987 over the period of observationand therapy;

FIG. 5 b X-ray 52 days post-OP of the lesion on the distal femur;

FIG. 5 c X-ray 96 days post-OP from the regressing lesion and the callusformation.

DETAILED DESCRIPTION OF THE INVENTION

The subject of the invention is thus a medicament for the therapy oftumor diseases, which contains as active ingredient a BSP specificbinder molecule such as an antibody, ligand or inhibitor. In oneembodiment, the binder molecule is an antibody or an aptamer on thebasis of RNA or DNA which recognises BSP in the presence of factor H.Particularly preferred binder molecules recognise specifically BSP fromtumor cells. The medicament can be reinforced with the followingsubstances: antibodies, ligands or inhibitors which interact withadhesion molecules, with membrane-associated proteases, or withreceptors which mediate chemotaxis such as for example the chemokinereceptors, and apotosis inducing substances such as preferablyantibodies, aptamers or proteins/peptides which are obtained fromnatural or artificial peptide banks. A specific protein (peptide)interaction preferably with non-specific molecules which are obtainedfrom, natural extracts, from synthetically or recombinantly producedbinding proteins, and from other peptide/protein banks, is however notsufficient to bring about the effect of apoptosis of tumor cells. Afterappropriate diagnosis, a specific therapy can be applied. Here,surprisingly, with the use of anti-BSP antibodies or binding proteinsthere is observed an accelerated occurrence of tumor cell death(apoptosis).

In particular, tumors which can be treated in this way are of the groupincluding breast, prostate, lung, kidney and thyroid tumors, and tumorsof the blood system, of the lymphatic system, of the heart andcirculatory system, of the nervous system, of the respiratory tract, ofthe digestive tract, of the endocrine system, the skin including adnexa,of the locomotory system, and of the urogenital tract including thekidneys.

The administering of the binding proteins and antibodies allows a newtherapy of tumor diseases on the basis of the BSP system andstrengthening with the inclusion of further tumor surface-associatedproteome clusters. The medicament is based on the employment ofBSP-specific antibodies, aptamers, ligands or inhibitors against theprimary or secondary tumor and dispersing metastases; that is, for thesuppression of cancer growth, inclusive of metastasisation. Themedicament in accordance with the invention is based on thedetermination that BSP acts, through autocrinic, paracrinic andendocrinic paths, via the disease specific constellation of the tumorcell proteomes on specific tumor cells. Primary and certain secondarytumors are controlled in their adhesion, migration and proliferationbehaviour. Through diagnostic determination of the locally increasedexpressed and regulated factors, and the presence of BSP, there isprovided the possibility of decisively suppressing or completelypreventing cancer growth, including tumor metastasation.

A further embodiment of the invention relates to the employment of theantibodies in accordance with the invention in pharmaceuticalcompositions for the treatment of tumors and metastases. The antibodiesin accordance with the invention, and their part structures orconjugates, can be applied through injection or via suppositories andcan bind and neutralise BSP, in the blood or in tissue fluid, freelycirculating or bound to factor H, Should there be a thus far non-provenprotective function of the factor H complex against the alternative pathof complement activation, this is inhibited and the tumor cells can beattacked by the immune system. Further, the angiogenic effect of BSP isinhibited.

For binding to the complex of factor H and BSP, the antibody mustrecognise epitopes of BSP which are not masked by the binding partner.The production of such antibodies has previously not been possible. Theinvention makes available such antibodies, because the antibodies aredirected against an isoform of the folded bone sialoprotein (BSP) andbind to epitopes which are formed only by a folded bone sialoproteinfrom tumor cells, the glycosylations of which are modified or incompleteor missing in the region of the amino acids 120 to 135 (with signalsequence), including the amino acid sequence TGLAA (SEQ ID NO: 15) orYTGLAA (SEQ ID NO: 16), in comparison to the normal bone sialoproteinfrom bones. Normally there cannot be attained specific antibodiesagainst post-translational or complex sugar structures on proteins,since such sugar structures are added in the same manner and form tomany different proteins. Correspondingly, antibodies react againstcertain sugar structures with many different proteins and are thenconsidered as a rule to be non-specific and of no value. This isdifferent with bone sialoprotein from tumor cells. The altered ormissing sugar structure brings about a different folding of the bonesialoprotein and creates new epitopes in which there are involved bothamino acids or peptide structure and also the many remaining sugarresidues. These epitopes are however characteristic for BSP fromdegenerate tumor cells.

Antibodies against these epitopes can be produced with a BSP, alteredchemically or naturally in its glycosylation, as antigen and ifapplicable through purification or absorption to the isoform of the boneBSP. Preferably the antibodies are produced with the employment of BSPfrom tumor cells as antigen. Since BSP from tumor cells can be isolatedin sufficient quantities only with difficulty, the geneticallyengineered expression of BSP modified in its glycosylation in tumorcells is the method of choice. It has also been found that some patientshave in the bone material BSP modified in its glycosylation. This meansthat these patients, mostly very old and suffering from serousosteoporosis, produce a BSP which at least in part is not normallyglycosylated. This BSP also is suitable in principle as an antigen forthe obtaining of the antibodies in accordance with the invention. Theisolation of the partially glycosylated isoform, which is comparable tothe tumor isoform or the BSP, can be carried out analogously todescribed procedures (Karmatschek M et al., Improved purification ofhuman bone sialoprotein and development of a homologousradioimmunoassay, in Clin. Chem. 1997, 43(11), 2076-82).

The antibodies can be produced in mice, guinea pigs, rabbits, dogs,goats, pigs, humans, donkeys or horses, but also in all mammals.Particularly preferred is the immunisation of birds, in particularchicken, since here due to the large evolutionary differences,antibodies against the tumor isoform of BSP can be obtained particularlyeasily. Further, the presence of IgY antibodies does not lead to anactivation of the complement system, which could be problematic due tothe possible binding between factor H and BSP. The antibodies inaccordance with the invention recognise the tumor isoform of BSP in thebonding with factor H.

Thus, subjects of the invention are isoforms of BSP, particularantibodies against the isoforms produced by tumors, and their use forantibody therapy or also for immune scintigraphy. As side effects, whichare brought about by anti-BSP antibodies, there come in question: directand indirect damage of the bones and dentin through activation of theimmune system against the bone matrix and bone and/or directdestruction, in the employment of conjugates of the antibody with cellpoisons or radio isotopes. Further, an immune scintigraphy isinconceivable with anti-BSP antibodies which bind to the bone matrix.The matrix would be radioactively marked and the localisation of tumorswould be impossible. The antibodies specific for human BSP are suitablein a particular manner for tumor therapy and localisation, since then donot bind or bind to only a slight extent to the bone matrix or to BSPproducing cells of the skeleton and the dentin.

In a particularly preferred application of the invention there are putto use for tumor therapy antibodies which are specific for tumor BSP andadditionally recognise BSP in the complex with factor H. Such antibodiesare made available by the invention. After application of such specificantibodies in tumor patients, free tumor BSP and tumor BSP bonded tofactor H, in the blood and in tissue fluid, is marked and therewith theprotection against complement activation removed. Thus, tumor cells arespecifically marked for destruction by the immune system (e.g. throughclassical activation of the complement cascade) and there are avoidedside effects such as e.g. through activation of the immune systemagainst the bone matrix or the dentin.

For the tumor therapy and immune scintigraphy made possible by means ofthe invention there can be employee by way of example polyclonalantibodies which can be produced immunisation of chickens withrecombinant BSP or BSP, modified in its glycosylation, Isolated frombones. The antibodies are then isolated in known manner from the eggyolk and purified by affinity chromatography.

In a further application of the invention, human polyclonal anti-BSPantibodies are isolated from the eggs of transgenic chickens havinghumanised immune system.

Likewise suitable are monoclonal antibodies from the mouse or thechicken, which fulfil the above-described conditions and which can beobtained by means of screening. In a specific application of the patentthere are employed for this purpose the monoclonal cell lines describedby way of example. Further suitable are Fab fragments obtained throughfragments of antibodies, e.g. proteolytically or by genetic engineering.

For the tumor therapy the above-described antibodies or antibodyfragments are further suitable, in conjugation with cell poisons andradioisotopes, for the direct destruction of tumor cells after bindingto BSP on the cell surface.

Particularly suitable are humanised poly- and monoclonal antibodieswhich recognise BSP in the complex with factor H and do not bind to BSPin the bone matrix. With the application of antibodies of the mouse andof the chicken there is however a particular therapeutic effect to beexpected through formation of human anti-mouse antibodies (HAMA) oranti-chicken antibodies (HACA). HAMAs and HACAs can induce andstrengthen an immune response of the organism to the tumor antigen. Inthe determination of tumor markers there arise, however, interferenceswith the HAMAs and HACAs which disrupt in vitro measuring methods. Inthis manner there are produced falsely high measurement values for tumormarker. This appears after immune scintigraphy or immune therapy withappropriate antibodies, so that a correct tumor marker determination invitro can be effected only after absorption of the HAMAs or HACAs.

These effects can be suppressed through the employment of humanisedantibodies. Polyclonal humanised anti-BSP antibodies can for example beobtained by immunisation of transgenic chickens with BSP, for whichchickens in the embryonic stem cells the gene region for thechicken-specific Fc part of the immunoglobulin (IgY) is exchanged for ahuman specific one (U.S. Pat. No. 5,340,740; U.S. Pat. No. 5,656,479).The humanised antibodies are then deposited in eggs of the chickens andcan be isolated from the egg yolk (Mohammed S. M. et al., Deposition ofgenetically engineered human antibodies into the egg yolk of hens.Immunotechnology, 1998, 4:115-125).

For the production of humanised monoclonal antibodies there may beobtained hybridoma cells of the mouse or the chicken with suitableanti-BSP antibodies, in accordance with standard methods, and from thegenetic material contained in these cells humanised antibodies can bedeveloped through recombination (U.S. Pat. No. 5,585,089; U.S. Pat. No.5,565,332; U.S. Pat. No. 5,225,539; U.S. Pat. No. 5,693,761; U.S. Pat.No. 5,585,089; U.S. Pat. No. 5,530,101).

The BSP can be put to use with the entire sequence SEQ ID No. 1 or thepart sequence ID No. 2, in its entirety or with its specific epitopesfor the generation of antibodies.

Preferred BSP fragments for the production of specific antibodies are:

X-Y T GLAAIQLPKKAGD-Z SEQ ID NO: 1 SEQ ID NO 2:X-FSMKNLHRRVKIEDSEENGVFKYRPRYYLYKHAYFYPHLKRFPVQGSS  DSSEENGDDSSEEEEEEEETSNEGENNEESNEDEDSEAENTTLSATTL   GYGEDATPGTGY TGLAAIQLPKKAGDITNKATKEKESDEEEEEEEEGN  ENEESEAEVDENEQGINGTSTNSTEAENGNGSSGVDNGEEGEEESVTG  ANAEGTTETGGQGKGTSKTTTSPNGGFEPTTPPQVYRTTSPPFGKTTT  VEYEGEYEYTYDNGYEIYESENGEPRGDNYRAYEGEYSYFKGQGYDGY   DGQNYYHHQ-Zwherein the marked T is not or is incompletely glycosylated, or isglycosylated in another form, and X and Z stand for amino acid orpeptide residues of up to 30 amino acids. In SEQ ID No. 2 the followingvariations may be present: at position 179 Gly→Val; position 252Val→Ala; position 254 Glu→Asp; position 279 Asp→Gly.

For the production of the antibodies the peptides, which are normallynot immunogenic, are coupled to the carrier protein KLH (keyhole limpethemocyanin). This coupling can be effected through NBS(N-maleimidbenzoyl-N-hydroxysuccinimide ester via a cysteine addedterminally in the peptide, or directly by means of carbodiimide.

The antibodies are obtained with conventional processes throughimmunisation preferably of chickens, rabbits, mice, guinea pigs etc.There can be put to use also molecular biological processes such as therecombinant production of the antibodies. The antibodies are thenpurified and galenically prepared. There may also due put to use cellpreparations, cell extracts and in particular membrane isolates fromover-expressing artificially transfected BSP expressing cells for thegeneration of specific antibodies.

The medicaments in accordance with the invention can be administered insuitable galenical application forms, in particular lyophilised form,taken up by mannite or similar sugars, in sterile ampoules fordissolving in physiological salt solution and/or infusion solution forrepeated individual injection and/or continuous infusion in quantitiesfrom 300 mg to 30 mg pure antibody or BSP ligand per therapy unit.Preferably the medicament in accordance with the invention is put to usein a galenical application form in which the medicament is administeredsystemically or locally in biocompatible microspheres and via aerosol,intravenous or subcutaneous application.

With various routine procedures it is possible to determine that thetumor cells, upon administration of agonists, which bind to thecorresponding proteome molecules, react in an anti-apoptotic, adhesive,mitotic and chemotactic manner. The restriction of its conservation,adhesion, mitotis or migration brought a bout through prior incubationwith antagonists or antibodies.

In the employment of highly purified antibodies against BSP in cellcultures of BSP-expressing tumor cell lines could be determined that inthe case of in vitro models these are in a position to bring about theapoptisis of tumor cells. If one cultivates cell lines or removed tumorcells with the employment of conventional cell culture processes, theirsurvival time in vitro is strongly reduced through the addition of BSPantibodies, if BSP can be detected on their corresponding cell surface.Thereby there can be observed apoptosis of a great number of thesecultivated cells. Also in the case of in vivo models one can,surprisingly, also determine a tumor cell regression through apoptosis.

Further, in experiments in the cell culture with tumor cells whichexpress BSP, the use of specific BSP antibodies can initiate complementmediated cell lysis and also cellular mediated tumor cell lysis.

Since hairless mice and hairless rats have a deficient immune system,metastasising behaviour can investigated in a host body in a hairlessmouse/rat model without the known immune reaction between species takingplace and leading to rejection of the foreign cells. Hairless mice areinoculated, in a manner known per se, with tumor cells or tumor celllines the BSP expression of which had been determined and the occurrenceof metastasisation due to these cells monitored upon treatment with BSPantibodies and upon treatment with BSP ligands. Thereby it issurprisingly found that in the case of the BSP positive tumors found theformation of metastases is significantly restricted or prevented,because the administration of antibodies leads to a modulation of thetumor growth. Surprisingly it is likewise found that the preparationsanalysed by means of immunohistochemistry show a specific distributionof BSP and other tumor surface associated proteome clusters in the tumorand tissue surrounding the tumor. Through this, further possibilitiesfor purposive attack have been recognised.

There is provided a broadening of the therapeutic concept throughadditionally employing antagonists in particular directed againstfurther clusters of the tumor cell surface proteomes. A reinforcing ofthis effect can attained through a combination of BSP antibodies withantibodies, ligands or inhibitors which interact with (1) adhesionmolecules, (2) membrane-associated proteases or (3) receptors whichmediate chemotaxis, such as for example chemokine receptors, and (4)apoptosis inducing substances such as preferably antibodies orproteins/peptides which are obtained from natural or artificial peptidebanks.

In order to confirm these findings, tumor cell lines can also be stablytransfected with BSP. After injection of these cells (in the case ofwhich BSP is over-expressed) into animals, such tumor cells settlepreferentially in the bone matrix. Such modified cells therewith form inparticular metastases in the bone tissue, on the basis of which thetherapeutic principle can likewise be demonstrated.

The invention will be described in more detail below with reference toexamples:

EXAMPLE 1 Characterisation of Tumor and Bone Specific BSP Isoforms inWestern Blot

Serum free supernatants of the human osteosarcoma cell lines UMR-108,MHH-ES 1 and of the breast cancer cell line MCF-7 (oestrogen receptorpositive) and also of human BSP K-BSP) purified from bones was separatedby means of SDS-PAGE on a 10% gel under reducing and denaturingconditions and electrophoretically transferred to nitrocellulose. Themembrane was incubated with the monoclonal mouse antibody. The detectionof BSP was effected via an anti-mouse antibody of the goat coupled toperoxidase, and chemoluminescence detection on an X-ray film. The resultis shown in FIG. 1. Molecular weight and path of the markers areindicated on the left side. The single and double arrowheads show thedifferent behaviour of the bone/osteosarcoma BSP and MCF-7 BSP. Thelatter contains additionally a high molecular weight band (triple arrow)which is absent in the other tracks. BSP from one tumor cell line thushas a significantly higher molecular weight than BSP from bone and fromosteosarcoma cell lines, whereby beyond this a second isoform with evenhigher molecular weight can be observed.

EXAMPLE 2 Production of Polyclonal Antibodies by Means of Immunisationof Chickens with Bone BSP and BSP Peptide Part Structures

Chickens and rabbits were immunised with BSP which was isolated frompatients in accordance with the procedure described by Karmatschek etal. (1997).

From the egg yolks and the sera, polyclonal immunoglobulins wereisolated and tested for binding against various peptide part structuresof BSP in an ELISA process. Table 1 shows the results of this epitopemapping. Thereby, peptide part structures of the overall 317 amino acidslong peptide sequence of preproBSP (including leader sequences) werechemically synthesised and bound to a microtitration plate and theantibodies incubated on the plate. The test for binding was effectedafter incubation with a conjugate of peroxidase with anti-IgYimmunoglobulins or anti-rabbit-IgG immunoglobulins and subsequent enzymereaction through transformation of a chromogene as substrate.

TABLE 1 Epitope mapping of the obtained anti-BSP IgG and IgY Position ofthe peptide part structure in the BSP ELISA Reaction strength (Positionincl. Leader) Amino acid sequence IgY Rabbit IgG 112-123 (SEQ ID NO: 3)LeuGlyTyrGlyGluAspAlaThrProGlyThrGly − ? 216-227 (SEQ ID NO: 4)GluThrGlyGlyGlnGlyLysGlyThrSerLysThr − ? 300-311 (SEQ ID NO: 5)PheLysGlyGlnGlyTyrAspGlyTyrAspGlyGln − ? 130-144 (SEQ ID NO: 6)IleGlnLeuProLysLysAlaGlyAspIleThrAsnLysAlaThr +/− + 124-138 (SEQ ID NO:7) TyrThrGlyLeuAlaAlaIleGlnLeuProLysLysAlaGlyAsp − ++ 137-151 (SEQ IDNO: 8) GlyAspIleThrAsnLysAlaThrLysGluLysGluLysGlu − + SerAspGlu 280-317(SEQ ID NO: 9) SerGluAsnGlyGluProArgGlyAspAsnTyrArgAlaTyr ++ +GluAspGluTyrSerTyrPheLysGlyGlnGlyTyrAspGlyTyrAspGlyGlnAsnTyrTyrHisHisGln Human bone BSP +++ +++

The results show that the obtained chicken antibodies preferentiallybind to the C-terminal sequence of BSP, whereas the rabbit antibodiesbind over a greater region.

Further, polyclonal antibodies (A0001) were obtained by means ofimmunisation of rabbits with the peptide structureTyrThrGlyLeuAlaAlalleGlnLeuProLysLysAlaGlyAsp (position 124-138) (SEQ IDNO: 7) of BSP which preferentially react to this peptide part structure,but also specifically with human bone BSP.

Polyclonal antibodies (AK_tBSP), however, which were obtained throughimmunisation of rabbits with the peptide part structures ThrGlyLeuAlaAla(position 125-130) (SEQ ID NO: 15), for example TyrThrGlyLeuAlaAla(position 124-130) (SEQ ID NO: 16), that is after coupling to bovinethyreoglobulin as carrier, react with the synthetic peptide partstructure, but not with human bone BSP. These antibodies, surprisingly,recognise exclusively BSP from tumor cells.

For the investigations there were further employed the polyclonalantibodies A002 (obtained from L. W. Fisher) and A003 (obtained from Dr.van Ryden). These antibodies were obtained after immunisation with thepeptide part structures

Application No. 10/480,465

(SEQ ID NO: 11) TyrGluSerGluAsnGlyGluProArgGlyAspAsnTyrArgAlaTyrGluAsp (A002) Or (SEQ ID NO: 12)  LeuLysArgPheProValGlnGlyGly.

The former peptide originated from the C-terminus of the BSP (positions278-295) and contains the RGD (ArgGlyAsp) recognition sequence of theBSP for receptors of the integrin type. The latter peptide orginatedfrom the N-terminus of the BSP primary structure. Also these peptidespreferentially recognised the respective part structures and reactedspecifically with human bone BSP.

EXAMPLE 3 Obtaining of Recombinant BSP from Breast Cancer Cells asAntigen

From the plasmid B6-5g (Fisher L. W. et al., Human bone sialoprotein.Deduced protein sequence and chromosomal localisation, in J. Biol.Chem., 1990, 265(4), 2347-51) the complete cDNA for human BSP (withoutsignal peptide) was amplified by means of PCR and cloned in the episomaleucaryotic expression vector pCEP-Pu (Kohfeldt E et al., Properties ofthe extracellular calcium binding module of the proteoglycan testican,in FEBS Lets. 1997, 414(3) 557-61). The primers were as follows:

Nhe I BSP (sense) (SEQ ID NO: 13)5′ GCCCGCTAGCCTTCTCAATGAAAAATTTGCATCG-3′ Not I BSP (antisense): (SEQ IDNO: 14) 5′-CAATGACTGCGGCCGCTCACTGGTGGTGGTAGTAATTC-3″

The Nhe I and Not I slicing sites introduced with the primers werenecessary for the cloning in the expression vector PCEP-PU. This vectoris moreover, for facilitating the protein purification, provided at the5′-ends of the multiple cloning sites with various tags (e.g. His, Myc,G8T). These tags can be detached after purification of the protein witha protease (e.g. factor X or enterokinase). That the correct readingframe was kept to was checked by means of sequencing.

The expression constructs were introduced by means of liposome mediatedstable transfection (FUGENE™ transfection reagent of the company Roche)inter alia into the following human cell lines:

-   -   the embryonic kidney cell line EBNA-293    -   the osteosarcoma cell lines SAOS-2 and MG-63    -   the human breast cancer cell line MCF-7.

A recombinant expression was obtained only in MCF-7 and EBNA-293 cells(see FIG. 2). The osteosarcome cell lines did not express even afterrepeated transfection attempts.

EXAMPLE 4 Analysis of the Glycosylation of Recombinant BSP fromDegenerate Cells and Bone BSP

Transient cells were cultivated, 48 hours after transfection, for twodays in serum-free medium. So that the proteins in the FCS did not makemore difficult the purification of the recombinant BSP, BSP expressingcells were, after attainment of confluence, cultivated under serum freeconditions. Under these conditions only EBNA-293 cells could survivelonger than 2 to 4. The expression of the recombinant BSP was monitoredthrough SDS-PAGE and immunoblots.

The investigation of serum-free cell culture supernatants yielded withall these cell lines a positive signal in the Western Blot, both withreference to BSP and also the presence of the various tags.

2.5 liter serum-free culture supernatant of the transfected MCF-7 cellline was purified via a Sepharose™ column and there was obtainedtherefrom 250 μg homogeneous His-myc-EK-BSP. The so purified expressionproduct was partially glyglosylated, however had no glycosylation atthreonin 125, that is the threonin in the BSP sequence YT¹²⁵LPAA (SEQ IDNO: 17).

For the glycoanalysis the N-glycanes were enzymatically separated fromthe recombinant BSP (rBSP) or the bone BSP with the peptideN-glycosidase F (PNGase F, Roche). The enzyme brought about a cataylyticsplitting of all N-glycane types from the asparagines. For thedigestion, 20 to 200 μg BSP was precipitated with ethanol and theprecipitant pellet incubated in 1% SDS, β-mercaptoethanol, 0.1 M EDTAfor 30 minutes at room temperature with an excess of enzyme. Therefollowed a digestion with N-glycosidase F overnight at 37° C. Forde-salting the N-glycane solution the digestion was given via a 150 mgcarbon column (carbograph SPE, Alltech) and the N-glycanes eluted with25% aCN in 0.05% TFA.

The O-glycanes were sliced from the BSP by means of water-freehydrazinolysis using a kit (Oglycan release kit, Glyco). For thispurpose, approximately 200 μg salt free BSP was lyophilised for 24hours, had 50 μl hydrazine reagent added thereto under argon protectivegas, dissolved and incubated for 5 hours at 60° C. The hydrazine wasdrawn off under vacuum. There followed a Re—N-acetylisation of theN-acetyl groups with acetic acid anhydrid.

The N— and O-glycanes were marked with the fluorescence dye2-aminobenzamide (Fluka) and the 2-AB marked oligosaccharides digestedsequentially with specific terminal glycosidases and analysed by meansof MALDI-TOF mass spectrometry.

Discussion of the Analysis

The amino acid sequence of human BSP contains four potentialN-glycosylation sites at the positions 88 (NTT), 161 (NGT), 166 (NST)und 174 (NGS).

For O-glycosylation there is known no comparable consensus sequence. Allidentified N-glycane structures could be found both on the BSP isolatedfrom bones and on the recombinant EBNA-293 BSP. There were howeverdifferences in the percentage proportion of the respective structures inthe total N-glycanes. Thus, the main proportion of the BSP N-glycanes inbones was of triantenary structures (58%) and in the EBNA cell line oftetraantenary structures (48%).

For localisation of the O-glycosylation sites of recombinant BSP, theO-glycanes were removed by means of sequential digestion of the proteinwith neuraminidase, β-galactosidase and β-N-actylhexosaminidase, down tothe core-GaINAc. The partially deglycosylated protein was then split bytreatment with trypsin and V8 protease into peptide fragments. By meansof MALDA-TOF mass spectrometry the masses of the peptides weredetermined and a part of the peptides sequenced by means ofPSD-MALDI-TOF mass spectrometry. With this method, eight O-glycosylationsites of the recombinant BSP could be determined, 5 on the peptide211-229 (of SEQ ID NO: 2) (TTTSP . . . QVYR) and a maximum of 3 on thepeptide between AS 120 and AS 135 having the sequence TGLAA (SEQ ID NO:15). Of these, in the recombinant BSP, the threonines in the sequenceDATPGTG (SEQ ID NO: 18) are O-glycosylated. With bone BSP there waseffected a third O-glycosylation. With recombinant BSP no thirdglycosylation site is present. Probably, this gylcosylation site lies onthe TGLAA-BSP part structure.

EXAMPLE 5 Production of Anti-BSP IgY from Egg Yolks

For the purification of greater quantities of anti-BSP IgY for therapyand immune scintigraphy there are described various processes. Theprocess of Akita and Nakai (Akita E. M. et al., Comparison of fourpurification methods for the production of immunoglobulins from eggslaid by hens immunised with an enterotoxigenetic E. coli strain, in JImmunol Methods. 1993, 160(2), 207-14) is preferentially used.

For the egg production there is used a highly productive species such as“Lohmann White” or “Lohmann Brown” with a productivity of 4.5 eggs perweek and a production of over 10 mg specific IgY per yolk. Theimmunisation was effected with BSP antigen isolated from human bones, orrecombinant, in Freund's Adjuvant, whereby after a basic immunisationwith ca. 0.1 mg BSP, booster infections where given every six weeks.Normally ca. 30% of these chickens do not react to the immunisation. Theeggs were externally disinfected with peracetic acid, then broken andyolk separated from egg white. The yolks were then whisked with 5 to 10times volume ice cold distilled water between pH 5 and 5.2 and incubatedat 2 to 5° C. over 2 to 6 hours. Thereby there sediments out the yolkgranulata which are substantially of lipoproteins. The aqueoussupernatant was then filtered clear through filter paper (e.g. WhatmanNo. 1).

From his supernatant, the anti-BSP IgY can be homogenously purifieddirectly or via affinity chromatography. There was chemically covalentlybonded, through a Sepharose 4B column, activated with cyanogen bromide,BSP isolated from human bones or from culture supernatants ofrecombinant human cell lines. For bonding 1 g IgY there is needed 0.5 gimmobilised BSP (covalently bonded to ca. 5 ml Sepharose™).

The bonded IgY is eluted via an acid gradient and thereafter thesolution neutralised. This solution must then be desalted and theantibodies concentrated, which is possible on an large scale in thecrossflow method (e.g. Amicon™, spiral filter SY 100 with a yield of100,000 Dalton).

EXAMPLE 6 Isolation of Anti-BSP IgY which is Bonded to the BSP Factor HComplex

The slight reaction of the polyclonal chicken antibody with BSP in thebone matrix can be excluded through selection of those antibodies whichreact with BSP in the complex with factor H. For this purpose there ischemically covalently bonded through cyanogen bromide activatedSepharose 4B either factor H or BSP isolated from bones or geneticallyengineered, and thereafter so much BSP or factor H applied to the columnand bonded that all ligands in the matrix are complexed with thepartner. Filtered yolk extract is then applied to this affinity columnand as in Example 4 there is now obtained that fraction of antibodieswhich specifically bonds to the free epitope in the BSP-factor Hcomplex.

EXAMPLE 7 Production of Human anti-BSP Antibodies in Transgenic Chickens

Anti-BSP IgY has in human therapy or diagnosis some weaknesses. Someside effects such as foreign protein reactions are to be expected andthe biological half-life amounts in comparison to human antibodies onlyto 12 to 24 hours. IgY does not activate the complement system.

Human antibodies against BSP can be produced in particular transgenicchickens, in which by means of gene targeting the constant region foravian immunoglobulin in the genes responsible for antibody formation hasbeen exchanged by the constant region for human immunoglobulin. Suitablechicken stem cells and vector systems are described in U.S. Pat. Nos.5,340,740, 5,656,479 and 5,464,764. After immunisation with BSP, suchchickens react with the production of human antibodies in the egg.

EXAMPLE 8 Immunblot Analysis of the Expression of BSP in Human BreastCancer Cell Lines

The tumor cell lines MDA-MB-231 (breast cancer cell line, oestrogenreceptor negative) MCF-7 (breast cancer cell line, oestrogen receptorpositive) and T-47-D (breast cancer cell line, oestrogen receptorpositive) were extracted with immune precipitation buffer and BSPprecipitated with the polyclonal antibody mixture A0001 of rabbitsagainst human BSP. The precipitates were applied, after denaturing, toSDS gels, the electrophoresis was carried out and the proteinstransferred to nitrocellulose membranes. Thereafter there followed animmune colouring with the anti-BSP rabbit antiserum A001 and amonoclonal mouse-anti-BSP antibody (BSP 1.2), whereby there was employedas second antibody peroxydase conjugates of antibodies of the goatagainst rabbit IgG and against mouse IgG. In both blots A and B thebands of the immune precipitated BSP can be clearly recognized at 70000Dalton.

In order to show the presence or absence of BSP on the cell surface oftumor cells, the cell surfaces of the breast cancer cell linesMDA-MB-231 and MCF-7 were biotinylated, extracted with immuneprecipitation buffer and BSP precipitated with the polyclonal antibodymixture A0001 of the rabbit against human BSP. The precipitates were,after denaturing, applied to SDS gels, the electrophoresis carried outand the proteins applied to a nitrocellulose membrane. Biotinylatedproteins on this membrane were then demonstrated with a conjugate ofperoxydase and streptavidine with the ECL system (Amersham).

Human breast cancer cells of the lines T-47-D and MDA-MB-231 were markedimmunofluorescently, both with and without prior permeablisation, withan anti-pig-BSP antibody from rabbit and an anti-rabbit antibody of thegoat conjugated with fluorescene, Fluorescently marked BSP can berecognised in both cell lines after permeablisation. Only in the T-47-Dcells could BSP be demonstrated by immunofluorescence also withoutpermeabalisation.

EXAMPLE 9 Detection of BSP Expression in Tumor Cells Via RT-PCP

From the tumor cell lines MDA-MB-231 (breast cancer cell line, oestrogenreceptor negative), MCF-7 (breast cancer cell line, oestrogen receptorpositive) and T-47-D (breast cancer cell line, oestrogen receptorpositive, and human fibroblasts (HGF) as control cells, there wasisolated mRNA, by reverse transcriptase the complementary cDNA wasproduced, and the BSP-cDNA amplified by means of PCR with BSP specificprimers. The expression of BSP-mRNA was particularly high in the breastcancer cell line MCF-7, slight in the case of the MDA-MB-231 and T-47-Dcells and not detectable in the control cell line.

EXAMPLE 10 Production of Humanised Monoclonal Antibodies

The monoclonal antibody BSP 1.2 can, due to its specific binding totumor BSP, be put to use for the therapy of primary tumors andmetastases. Thereby, the antibody binds on BSP on the cell surface ofcertain tumor cells and stimulates the immune system to destroy thecells, e.g. via the activation of the complement cascade. Similarly,there can be put to use also the polyclonal or monoclonal anti-BSP IgYfor therapy. When this antibody is used, the human immune system reactswith the formation of its own antibodies—human anti-mouse-IgG antibodies(HAMAs) or human anti-chicken-IgY; antibodies (HACAs). HAMAs and HACAscan induce or strengthen an immune response of the organism to the tumorantigen. In the determination of tumor markers there arises, however,interferences with the HAMAs and HACAs which disrupt in vitromeasurement methods. In this way there arises falsely high measurementvalues for tumor marker.

Thus, humanised monoclonal antibodies are particularly suitable for thetherapy and immune scintigraphy. A plurality of methods have beendescribed how one derives appropriately humanised antibodies from thehybridoma cell lines, which produce monoclonal anti-BSP antibodies.

EXAMPLE 11 Conjugates of Anti-BSP Antibodies with Cell Poisons andRadioisotopes

In a further application of the invention here may be chemicallycovalently bonded with the anti-BSP antibodies or their Fab fragmentscell poisons and radioisotopes. Antibodies marked with radioisotopessuch as iodine 125 or iodine 131 are suitable with the application ofsmaller quantities for tumor localisation via immune scintigraphy andwith the application of greater quantities or the direct destruction ofthe tumors. Such chemical conjugates can be produced for example byiodisation of the antibody with iodine 125 or 131 (Garvey, J. S et al.,Methods in Immunology. 3^(rd) ed., W. A. Benjamin Publ., 177, 171-182).An overview of suitable methods for radio immune therapy and immunescintigraphy is found in Vuillez, Radioimmunotargeting: diagnosis andtherapeutic use, in Bull Cancer. 2000, 87(11), 813-27.

EXAMPLE 12 Therapy of Tumors with Expression of BSP on the Cell Surface

It was first determined from biopsy material whether BSP was expressedon the surface of the tumor cells. Patients for whom BSP can be detectedon the surface of the tumor cells can be considered for therapy withanti-BSP antibodies of the chicken, the mouse, the correspondinglyhumanised antibodies and with conjugates of these antibodies with cellpoisons or radioisotopes.

The treatment of tumors with therapeutic antibodies which are directedagainst tumor markers expressed on the cell surface is state of the art.Thus, with the humanised antibody herceptin, against the receptor forthe human epithelial growth factor, breast cancer can be successfullytreated, even in the metastasising form, in ca. 25% of those affected(Hotaling TE et al., The humanized anti-HER2 antibody rhuMAb HER2mediates antibody dependent cell-mediated cytotoxicity via FcgR III[abstract]. Proc Annu Meet Am Assoc Cancer Res 1996; 37:47; Pegram M Det al., Antibody dependent cell-mediated cytotoxicity in breast cancerpatients in Phase III clinical trials of a humanized anti-HER2 antibody[abstract]. Proc Am Assoc Cancer Res 1997; 38:602.

Similarly as with herceptin, the appropriate anti-BSP antibody can beapplied as an infusion, e.g. as a 90 minute infusion in the firstapplication and later as a 30 minute infusion. The frequency of theinfusions and the quantity of the antibodies are determined inaccordance with the half-life of the antibodies in the blood (ca. 6 dayswith a humanised antibody and less than 24 hours with a chickenantibody) and the body weight.

EXAMPLE 13 Therapy of Tumors by Means of Neutralisation of Free BSP, notBonded to Cells, and of the BSP-factor H Complex

With the methods described above it was determined that the tumor cellsof the patient express BSP which cannot be detected on the cell surface.In the case of these tumors it can be assumed that the cells give outBSP into the blood or the tissue fluid and e.g. through binding offactor H use this for the inactivation of the alternative path of thecomplement cascade or for migration into bone tissues. A furtherpossible indicator for this tumor type are increased concentrations ofthe BSP in the blood serum (>20 ng/mL serum). In these cases anti-BSPantibodies can be out to use for the neutralisation of the free tumorBSP or the tumor BSP in complexes with factor H. The dose can then beset with regard to the quantity of the BSP present free in the serum andin the tissue fluid. For the therapy, there can be considered anti-BSPantibodies of the chicken, of the mouse and humanised anti-BSPantibodies, which can recognize the free BSP epitope in the complex withfactor H. There can also be considered Fab fragments of theseantibodies, which can be prepared in accordance with a standardprocedure by means of proteolytic digestion (Garvey, J. S et al.,Methods in Immunology. 3^(rd) ed., W. A. Benjamin Publ., 1977, 256-266).Also genetically engineered Fab fragments, derived from the aboveanti-BSP antibodies, come into consideration for such a therapy.

The invention thus makes available antibodies against the human bonesialoprotein (hBSP) which bind specifically only epitopes of hBSP oftumor cells, since tumor hBSP contains no post-translationalO-glycosylation in the region of the amino acids 120 to 135 (SWISSPROT:SIAL_HUMAN, Acc. No. P21815, without signal sequence) containing theamino acids TGLAA (SEQ ID NO: 15). Differently from the normal hBSP frombones. The antibodies can recognize tumorgenic serum hBSP in the complexwith the complement factor H and thus constitute a diagnostic andtherapeutically valuable instrument.

EXAMPLE 14 Production of Specific Antibodies Against BSP or OtherClusters of the Tumor Cell Surface Proteomes

For the production of specific antibodies against the said proteins ithas surprisingly been found that along with the use of completemolecules also specific amino acid sequences of epitopes are suitableparticularly for the immunisation when the synthesised peptides arecoupled to carrier molecules in accordance with the usual methods andinjected into mice. Further, also multiple antigenic peptides (sequencessee above) are suitable which are bonded to larger molecules by means oflysine, or BSP transfected cell lines, in order to produce theseantibodies. As a further method, the employment of immunogenes of stabletransfected BSP-expressing cells has proved itself surprisingly well,whereby membrane isolates, cell extracts with complete or fragmentedreceptors or also lyophilisated overall preparations are used.

Mice (Type NZW X NZB) were put to use for production of monoclonalantibodies, which was carried out with the routine methods ofImmundiagnostik AG and IPF PharmaCeuticals GmbH. The antibodiesmonitored by means of Western Blot and ELISA can, after high levelpurification, be put to use for the mentioned diagnostic and therapeuticpurposes.

EXAMPLE 15 Initiation of Apoptosis by Means of BSP Specific Antibodiesin BSP Expressing Cell Lines

Expression analysis of various cell lines have inter alia shown thatprostate tumor cell lines and also breast tumor cell lines express BSP.This expression was carried out on the mRNA level by means of RT-PCR andon the protein level by means of Western Blot and FACS analysis. Atreatment of these BSP expressing tumor cell lines with BSP specificantibodies has lead in cultivated cells to programmed cell death, whichchould be detected inter alia by means of through-flow cytometry.

EXAMPLE 16 Reduction of Bone Metastases in the Animal Model

After the application of BSP expressing tumor cell lines in immunedeficient hairless mice/hairless rats bone metastasisation regularlyoccurs. With the simultaneous administration of the BSP specificantibody there occurred, surprisingly, a significant reduction of theformation of manifest bone metastases, which could be proven throughhistological analysis of the tissue.

1. Material and Methodology

Our animal model involved injection of breast cancer cells andsubsequent therapy of the lytic lesions arising with anti-BSPantibodies, which represented a mixture of polyclonal IgY antibodies ofthe chicken with predominant specificity for human BSP from tumor cells,which also quantitatively bind hBSP in human serum in the presence offactor H, and which mainly bind to an epitope in the region of aminoacids 120 to 135 of the hBSP, whereby the post-translationalglycolsylation in this region is in the case of hBSP from tumor cellsaltered in comparison with natural BSP from bones.

There were injected MDA-MB 231-cells (ATCC, HTB-26), which in a previousstudy with hairless mice have been applied intracardially (T. A. Guise,1997; PTH-rP and Bone metastases; American Cancer society). The cellline was obtained from a metastasising human adenocarcinoma; further ithas no oestrogen receptors. In our case, it was marked with greenfluorescent protein (GFP) which facilitated the identification of thecells in the histological preparation. As experimental animals therewere used hairless rats of an age from 6 to 8 weeks (RNU, Charles RiverBreeding, Sulzfeld, Germany) which have reduced immune competency, sothat the injected human cells are not recognised as such and combatted.Our preparatory investigations with various cell quantities, with maleand female animals, showed that in the case of male rats the metastaseswere visible in the form of lytic lesions following injection of 10⁵MDA-MB 231 cells, after about one month.

The cells (1×10⁵ in PBS puffer) were injected intraarterially into theA. femoralis (0.2 ml; n=8). For this purpose a side branch of thisvessel was cannulised and after injection bound off, in order to preventexit of the introduced cells. The animal compensated the loss of thevessel by means of collateral formation without difficulty. The cancercells then entered with the blood into the fine branchings of thesupplying branches of the femur, tibia and fibula. Here, in the terminalflow path, there takes place extravasation and subsequent adhesion ofthe cells to the bone matrix. It is probably here also that theinteraction with BSP takes place.

The subsequent monitoring of metastases growth was effected on a ten daybasis with conventional X-ray images, anterior-posterior andposterior-anterior, with the animal anaesthatised with ether. Anapproximate quantification was carried out by means or measurement oflesion in terms of the length and width of its extent.

After two positive X-ray checks, the animals were treated withanti-BSP-Ak or standard. The therapy was effected with the describedanimals once per week subcutaneously in a concentration of 10 mg/kg bodyweight.

The follow-up observation of the animals was further effected by meansof computer tomography and histology (not yet completed). The CT alloweda three-dimensional reconstruction of the bone and of the defect, and anexact measurement of the lesion size. After the observation time theanimals were killed and investigated histologically. By means of themarking with GFP the cells are visible under UV light in the bonesection preparation. Further, histologically more direct information ofthe proceeding transformation of the bone tissue could be made.

2. Results

The results are documented by way of example in the accompanying seriesof FIGS. 4 and 5. The animals were observed after the operation for morethan three months by X-rays (Siemens Opti 150/30/50 HC-100). In thedevelopment of the curves attention is to be given to the fact that thearea indications of the lesion size in mm², indicated on the ordinate,are different for the two indicated animals. Overall, eight animals weretreated. Animals 987 and 988 see the Figures) were treated)subcutaneously with the anti-BSP-Ak (10 mg/kg) once per week. Theoverall duration of the treatment was ca. 50 days. The therapy wascommenced after 2 or 3 positive X-ray checks. In the case of animal 987this corresponds to day 46 after the operation, and in the case ofanimal 988 to the 32^(nd) postoperative day.

A rapid lesion size increase from the 24^(th) postoperative day was inthe case of animal 988 followed by a advancing lysis of the bone, whichfrom day 38 led to a fracture in the middle third of the tibia. Thistook place after therapy had begun (from day 32). First healingtendencies show themselves from day 52 in the form of callus formationat the fracture site, and also the lesions on the proximal tibia and adistal femur became smaller. The outer bounderies of the lesions wereinitially sharply defined, later during the healing increasingly lesssharply defined. The new formation of the bone took place starting fromthe outer perimeter towards the center of the lesion. From day 89 thelesion size could be quantified only with difficulty, since through theincreasing callus formation the exact edges could no longer berecognised. After the 126^(th) day one can speak of a completeremission, which is shown by the accompanying image (completedisappearance of the X-ray recognisable lytic leasion). Thethree-dimensional CT reconstructions (Siemens Somato Plus 4, VolumeZoom) show the changes of the tibia lesion after 31 and 80 days post OP.There can be seen a clear increase of bone tissue.

Animal 987 was, after three positive X-ray findings, also treated with10 mg/kg s.c. once per week from day 46. Here there appeared only ametastasis in the distal femur which already from the 89^(th) day (thatis after 42 days of treatment) was healed.

Other animals were, under the same conditions, treated up to five timesper week with 10 mg/kg s.c. and up to twice per week with 10 mg/kg i.v.There could be observed, however, an increase of size of the lesionduring and after completion of the treatment, if during the treatment animmune reaction against the injected BSP antibodies was induced.

For comparison, animals were treated twice per week withalkylphosphorcholine Er-PC₃ with 40 μMol/kg i.v. which at thisconcentration allowed primary breast carcinoma to regress (positivecontrol), but showed no effects in the case of bone metastases (Berger,M. R. et al., (1998) Erucylphosphocholine is the prototype of i.V.injectable alkylphosphocholines. Drugs of Today, 34 (Sppl. F), 73-82).Here, after the completion of the treatment, in the case of one animalno change was shown, by another even a deterioration of the situation(progression) in comparison to the beginning of treatment.

The subject of the present invention is thus a medicament containingantibodies or binding molecules such as aptamers against tumor specificBSP or other ligands for the same protein. Further, the employment ofthe proposed medicament application can be reinforced through the use ofthe following substances: antibodies, ligands or inhibitors whichinteract with adhesion molecules, membrane-associated proteases orreceptors which mediate chemotaxis, for example chemokin receptors, andapoptosis inducing substances such as preferably antibodies orproteins/peptides which may be obtained from natural or artificialpeptide banks. The medicament can be employed alone or in combinationwith the above-mentioned substances in particular for the therapy oftumor diseases, preferably their bone metastases.

The invention further relates to a method of therapy and the medical andcommercial use of the said antibodies against BSP or other ligands forthe same protein, or their combination with reinforcing antibodies orligands or inhibitors which interact with adheasion molecules,membrane-associated proteases or receptors which mediate chemotaxis,such as for example chemokin receptors, and apoptosis inducingsubstances such as preferably antibodies or proteins/peptides which canbe obtained from natural or artificial peptide banks, in order tosuppress the cancer growth inclusive of metastasisation. The method isbased on the determination that BSP can act on specific tumor cellsthrough the disease-specific constellation of the expression. Primaryand secondary tumors are, inter alia controlled in their migration andproliferation behaviour by BSP. From this there is provided thepossibility of decisively hindering or completely suppressing cancergrowth and tumor metastasisation by means of the said method/therapy.

1. A pharmaceutical composition for treatment of tumors and theirmetastases, which preferentially settle in bone tissue, comprising as anactive ingredient at least one antibody which specifically binds to bonesialoprotein in the presence of complement Factor H, at an epitopecomprising the amino acid sequence TGLAA (SEQ ID NO: 15) or YTGLAA (SEQID NO: 16) and a pharmaceutically acceptable carrier.
 2. Thepharmaceutical composition according to claim 1, wherein said at leastone antibody specifically binds bone sialoprotein from tumor cells anddoes not bind to bone sialoprotein from normal cells.
 3. Thepharmaceutical composition according to claim 1, wherein the antibodybinds epitopes which are only presented on human bone sialoprotein fromtumor cells, the post-translation glycosylation of which in the regionof amino acids 108 to 122 of SEQ ID NO: 2 containing the amino acidsTGLAA (SEQ ID NO: 15) is modified or incomplete in comparison withnormal bone sialoprotein from human bones.
 4. The pharmaceuticalcomposition according to claim 1, in which the active ingredient is anantibody that is an IgY antibody obtained from chicken.
 5. Thepharmaceutical composition according to claim 4, in which the IgYantibody from chicken is a human or humanised antibody.
 6. Thepharmaceutical composition according to claim 1, wherein the antibody isa bispecific antibody that comprises also an additional paratope that isspecific for epitopes of CD3.
 7. The pharmaceutical compositionaccording to claim 1, wherein said antibody is coupled with aradionuclide.
 8. The pharmaceutical composition according to claim 1,comprising additionally at least one antibody, ligand or inhibitor,selected from the group comprising adhesion molecules,membrane-associated proteases, receptors which mediate chemotaxis,chemokine receptors and apoptosis inducing substances.
 9. Apharmaceutical composition comprising as an active ingredient at leastone antibody which specifically binds to bone sialoprotein at an epitopecomprising the amino acid sequence TGLAA (SEQ ID NO: 15) or YTGLAA (SEQID NO: 16), in which the post-translation glycosylation of at least onethreonine in the region of amino acids 108 to 122 of SEQ ID NO: 2 isabsent.